Expression and characterization of PhzE from P. aeruginosa PAO1: aminodeoxyisochorismate synthase involved in pyocyanin and phenazine-1-carboxylate production

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• 作者：Justin E. Culbertson ; Michael D. Toney ; ^{mdtoney@ucdavis.edu}
• 关键词：AS ; anthranilate synthase ; SS ; salicylate synthase ; ADCS ; aminodeoxychorismate synthase ; IS ; isochorismate synthase ; CM ; chorismate mutase ; ADIC ; aminodeoxyisochorismate ; DHHA ; trans-2 ; 3-dihydro-3-hydroxyanthranilic acid ; GDH ; glutamate dehydrogenase ; LDH
• 刊名：Biochimica et Biophysica Acta - Proteins and Proteomics
• 出版年：2013
• 出版时间：January, 2013
• 年：2013
• 卷：1834
• 期：1
• 页码：240-246
• 全文大小：767 K

文摘

PhzE from Pseudomonas aeruginosa catalyzes the first step in the biosynthesis of phenazine-1-carboxylic acid, pyocyanin, and other phenazines, which are virulence factors for Pseudomonas species. The reaction catalyzed converts chorismate into aminodeoxyisochorismate using ammonia supplied by a glutamine amidotransferase domain. It has structural and sequence homology to other chorismate-utilizing enzymes such as anthranilate synthase, isochorismate synthase, aminodeoxychorismate synthase, and salicylate synthase. Like these enzymes, it is Mg^2 + dependent and catalyzes a similar S_N2"" nucleophilic substitution reaction. PhzE catalyzes the addition of ammonia to C2 of chorismate, as does anthranilate synthase, yet unlike anthranilate synthase it does not catalyze elimination of pyruvate from enzyme-bound aminodeoxyisochorismate. Herein, the cloning of the phzE gene, high level expression of active enzyme in E. coli, purification, and kinetic characterization of the enzyme is presented, including temperature and pH dependence. Steady-state kinetics give K_chorismate = 20 ¡À 4 ¦ÌM, K_Mg^2 + = 294 ¡À 22 ¦ÌM, K_L-gln = 11 ¡À 1 mM, and k_cat = 2.2 ¡À 0.2 s^? 1 for a random kinetic mechanism. PhzE can use NH₄⁺ as an alternative nucleophile, while Co^2 + and Mn^2 + are alternative divalent metals.